This proposal involves studies on the molecular mechanism of enterotoxin B production and its regulation in Staphylococcus aureus. Staphylococcal enterotoxins are exoproteins that are involved in its pathogenicity and are causative agents of staphylococcal food-poisoning. Genetic studies have suggested that the enterotoxin B gene (entB) may be part of a mobile genetic element. We have cloned the entB gene from S. aureus food-poisoning isolate, strain S6, and determined its nucleotide sequence. Subcloning and in vitro deletion experiments will be carried out in S. aureus using the cloned entB gene to identify the involvement of a positively-acting regulatory gene (entB) in toxin production. Northern hybridization analysis and nuclease S1 mapping experiments will be carried out to determine the transcriptional start and stop sites of the entB and entR (if any) genes. These results will also indicate if the entB gene is regulated at the level of transcription. There is a more than 100-fold difference between the amount of enterotoxin B produced by different S. aureus strains. We have cloned the entB gene from the high toxin-producer strain S6. The entB gene will also be cloned from two low-producer strains. The amount of toxin and entB mRNA produced by clones carrying the entB gene from high and low producer strains in the same host background will be compared by SDS-polyacrylamide gel electrophoresis followed by immunoblotting and Northern blot analysis. The nucleotide sequence of the promoter region of the entB gene from the low toxin-producer strains will be determined and compared with the corresponding sequence from strain S6. These experiments are likely to identify the molecular basis of the variation in the amount of toxin produced by various strains. A positively-acting element, agr, that regulates the production of exoproteins in S. aureus has been identified. The cloned entB gene will be transferred into an agr- strain to determine if the agr element also regulates enterotoxin B synthesis. Southern hybridization experiments will be carried out to determine if the entB gene is part of a discrete genetic element. Genetic and biochemical experiments will also be carried out to determine if the entB gene is part of a phage or a transposon. A successful completion of our studies could reveal the molecular mechanism of enterotoxin B production and its regulation in S. aureus.